Effect of egg turning and incubation time on carbonic anhydrase gene expression in the blastoderm of the Japanese quail (Coturnix c. japonica)

1. The gene expression of carbonic anhydrase, a key enzyme for the production of sub-embryonic fluid (SEF), was assessed in turned and unturned eggs of the Japanese quail. The plasma membrane-associated isoforms CAIV, CAIX, CAXII, CAXIV, and the cytoplasmic isoform CAII, were investigated in the extra-embryonic tissue of the blastoderm and in embryonic blood. 2. Eggs were incubated at 376C, c.60% RH, and turned hourly (90) or left unturned. From 48 to 96h of incubation mRNA was extracted from blastoderm tissue, reverse-transcribed to cDNA and quantified by real-time qPCR using gene-specific primers. Blood collected at 96h was processed identically. 3. Blastoderm CAIV gene expression increased with the period of incubation only in turned eggs, with maxima at 84 and 96h of incubation. Only very low levels were found in blood. 4. Blastoderm CAII gene expression was greatest at 48 and 54h of incubation, subsequently declining to much lower levels and unaffected by turning. Blood CAII gene expression was about 25-fold greater than in the blastoderm. 5. The expression of CAIX in the blastoderm was the highest of all isoforms, yet unaffected by turning. CAXII did not amplify and CAXIV was present at unquantifiable low levels. 6. It is concluded that only gene expression for CAIV is sensitive to egg turning, and that increased CAIV gene expression could account for the additional SEF mass found at 84 to 96h of incubation in embryos of turned eggs.